Initiation of reverse transcription occurs at the primer binding site (PBS), which is complementary to the 3' terminal 18 results nucleotides of the cellular tRNALys, 3. Studies from our laboratory have demon- strated that mutations within U5 and the PBS results in virus which stably utilizes tRNAHis or tRNAMet to initiate reverse transcription. Based on these studies, we conclude that the RNA genome is a major determinant for the selection of the tRNA used to initiate reverse transcription. Delineation of the parameters for the interaction between tRNALys,3 and the PBS then represents an ideal new target for therapeutics. To further define the interaction between U5-PBS and the tRNA, we will use a genetic approach to identify critical regions within U5 required for selection of the tRNA. The following specific aims are proposed: -Specific Aim 1: To establish the importance of three regions within US for maintenance of PBS complementary to tRNAMet or tRNAHis. We have used RNA fold programs to identify three RNA structures within the U5-PBS. Using defined mutants in these regions, we will delineate the importance in reverse transcription/viral replication. Parallel experiments will be performed with relevant mutants using viruses which infect primary cells. Specific Aim 2: To correlate that mutations in U5 disrupt the capacity to interact with the tRNA used to initiate reverse transcription. A unique RT/PCR method will be used to analyze initiation of reverse transcription. A complementation system will be used to analyze the effects that mutations in U5 have on the completion of reverse transcription. Specific Aim 3: To investigate whether the tRNA U5-PBS interaction occurs prior to release of virus. Nuclear and cytoplasmic extracts will be generated from cells transfected with wild type or mutant proviral genomes. The RT/PCR method will be used to determine if correct initiation of reverse transcription occurs in the presence of added viral proteins. The integration of our genetic approach with the chemical/enzymatic studies will provide a clear picture of the RNA-RNA interaction between U5-PBS-tRNA. From the modeling and structural analysis, we will refine our understanding of critical regions of the U5-PBS-tRNA interaction. Finally, our unique viral systems which utilize tRNAHis or tRNAMet to initiate reverse transcription will be used to test the specificity and effectiveness of new therapeutics designed to inhibit the tRNA-U5-PBS interaction of the wild type genome.